TABLE 1.

Detailed description of the studies that used carvacrol, included in the systematic review.

Model	Concentration/incubation time	Experimental methods for testing IC50 values	Results/targets			Conclusion	Authors (Year), Country
			Increase	Decrease	IC50
Monoterpene carvacrol
In vitro studies
CO25	1–150 μg/mL	MTT assay	p21N−ras	Tumor growth	60 μg/mL–24 h	Carvacrol has a cytotoxic effect and an antiproliferative effect	Zeytinoglu et al. (2003), Turkey
	24, 48, 72 h of incubation			DNA synthesis level
A549	100–1,000 μM	—	Apoptosis induction	Cell viability	—	Carvacrol may have an anticancer effect and be used as a drug substance to cure cancer	Koparal and Zeytinoglu (2003), Turkey
	24 h of incubation			Cell proliferation
HepG2	25–900 μmol	—	Cytotoxic effects	DNA damage level	—	HepG2 cells were slightly more sensitive to the effects	Horváthová et al. (2006), Slovakia
Caco-2	24 of incubation
Leiomyosarcoma	10–4,000 μM	Trypan Blue	Antiproliferative effects	Cell growth	90 μM–24 h	Carvacrol has anticarcinogenic, antiproliferative and antiplatelet properties	Karkabounas et al. (2006), Greece
	24 and 48 h of incubation				67 μM–48 h
K-562	200–1,000 μM	Trypan blue exclusion	Cytotoxic effects	DNA damage level	220 μM–24 h	Carvacrol has cytotoxic, antioxidant effects and has a protective action against DNA damage	Horvathova et al. (2007), Slovakia
	24 or 48 h of incubation
P-815	0.004–0.5% v/v	MTT assay	—	—	<0.004% v/v–48 h	Carvacrol is cytotoxic	Jaafari et al. (2007), Morocco
	48 h of incubation
HepG2	100–1,000 μM	Trypan blue exclusion	Cytotoxic effects	Cell proliferation	HepG2 - 350 μM–24 h	Carvacrol has antiproliferative and antioxidant effects	Slamenová et al. (2007), Slovakia
Caco-2	24 h of incubation				Caco-2 - 600 μM–24 h
MDA-MB 231	20–100 μM	MTT assay	Apoptosis induction	Cell growth	100 μM–48 h	Carvacrol can be a potent antitumor molecule against breast cancer metastatic cells	Arunasree, (2010), India
			Caspase activation	S-phase cells
	24 or 48 h of incubation		Sub-stage G0/G1	Mitochondrial membrane potential
			Cyt C	Bcl-2
			Bax
5RP7	0.0002–0.1 mg/mL	MTT assay and Trypan Blue exclusion	Cytotoxic effects	—	5RP7 - 0.04 mg/mL–24/48 h	Carvacrol promoted a cytotoxic effect, induced apoptosis and can be used in cancer therapy	Akalin and Incesu, (2011), Turkey
CO25	24 or 48 h of incubation		Apoptotic cells		CO25–0.1 mg/mL–24 h
					0.05 mg/mL–48 h
SiHa	25–500 μg/mL	MTT and LDH assay	Apoptosis induction	Cell proliferation	SiHa - 50 ± 3.89 mg/L	Carvacrol is a potent anticancer compound that exhibits cytotoxic effects and induces the inhibition of cell proliferation in both human cervical cancer cells	Mehdi et al. (2011), India
HeLa	48 h of incubation				HeLa - 50 ± 5.95 mg/L
HepG2	20–200 μg/mL	CellTiter-Blue® cell viability assay	Cytotoxic effects	Membrane damage	53.09 μg/mL	Carvacrol exhibits antioxidant activity and anticancer effects on cells	Özkan and Erdogan (2011), Turkey
	24 h of incubation		Antiproliferative effects	Cell viability
P-815	0.05–1.25 μM	MTT assay	Cytotoxic effects	Interruption of cell cycle progression in the S phase	P-815–0.067 μM	Carvacrol showed a cytotoxic effect in all strains tested	Jaafari et al. (2012), Morocco
CEM					CEM - 0.042 μM
K-562	48 h of incubation				K-562–0.067 μM
MCF-7					MCF-7 - 0.125 μM
MCF-7 gem					MCF-7 gem - 0.067 μM
DBTRG-05MG	200–1,000 μM	—	Generation of ROS	Cell viability	—	Carvacrol was cytotoxic and induced cell death in human glioblastoma cells	Liang and Lu, (2012), China
	24 h of incubation		Caspase-3
H1299	25–1800 μM	CellTiter-Blue® cell viability assay	MDA	Membrane and DNA damage	380 μM–24 h	Carvacrol exhibited cytotoxic and antioxidant effects	Ozkan and Erdogan (2012), Turkey
	24 and 48 h of incubation		8-OHdG		244 μM–48 h
B16-F10	Not reported	Trypan blue assay and MTT assay	Cytotoxic effects	Cell viability	550 μM	Carvacrol showed an antitumor effect with moderate cytotoxicity	Satooka and Kubo (2012), United States
	24 h of incubation			Relative melanogenesis
				Relative melanin cell
HepG2	0.05–0.4 mmol/L	MTT assay	p-p38	Cell viability	0.4 mmol/L–24 h	Carvacrol caused inhibition of cell proliferation, inhibition of tumor cell growth and induction of apoptosis	Yin et al. (2012), China
	24 h of incubation		MAPK	p-ERK 1/2
			Caspase-3	Bcl-2
OC2	200–1,000 μM	—	Generation of ROS	Cell viability	—	Carvacrol exhibited a cytotoxic effect and induced apoptosis in human oral cancer cells	Liang et al. (2013), China
	24 h of incubation		Caspase-3
MCF-7	140–450 μM	MTT and LDH assay	Caspase-3, -6 and -9	Cell viability	244.7 ± 0.71μM–48 h	Carvacrol induces cytotoxicity and apoptosis in MCF-7 cells and may be a potential chemotherapeutic agent against cancer	Al-Fatlawi and Ahmad (2014), India
	24 and 48 of incubation		Bax	Bcl-2
			p53
N2a	10–400 mg/L	—	TAC	—	—	Carvacrol has antioxidant and anticancer properties in N2a cells at concentrations of 200 and 400 mg/L	Aydın et al. (2014), Turkey
	24 h of incubation		TOS
Caco-2	100–2,500 μM	MTS assay	Apoptosis induction	Cell viability	460 ± 3.6 μM–24 h	Carvacrol exhibited cytotoxic effects and induction of apoptosis	Llana-Ruiz-Cabello et al. (2014), Spain
	24 and 48 h of incubation				343 ± 7.4 μM–48 h
HepG2	25–1,000 μM	Trypan Blue exclusion and MTT assay	Apoptosis induction	Cell growth	425 μM–24 h	Carvacrol can be used as an anti-tumor molecule against cancer cells	Melusova et al. (2014), Slovakia
	24 h of incubation			SsDNA breaks
				Oxidative DNA lesions
HepG2	100–600 μM	—	Cells in G1 phase	S-phase cells	—	Carvacrol caused induction of apoptosis and slowed cell division, resulting in cell death	Melušová et al. (2014), Slovakia
	24 h of incubation
U87	125–1,000 μM	MTT assay	Apoptosis induction	Cell viability	561.3 μM–24 h	Carvacrol has therapeutic potential for the treatment of glioblastomas by inhibiting TRPM7 channels	Chen et al. (2015), Canada
	24, 48 or 72 h of incubation		Caspase-3	Cell proliferation
				PI3K/Akt
				MAPK
				TRPM7
				MMP-2
HCT116	100–900 μmol/L	MTT assay	Apoptosis induction	Cell growth	HCT116–544.4 μmol/L–48 h	Carvacrol can be a promising natural product in the management colon cancer	Fan et al. (2015), China
LoVo	48 h of incubation			Cell migration and invasion
				Bcl-2
			Bax	MMP-2 and -9	LoVo - 530.2 μmol/L–48 h
				Cyclin B1
				p-ERK
			p-JNK	p-Akt
				PI3K/Akt
				Cell cycle stop in phase G2/M
AGS	0.01–6 mg/mL	MTT assay	Cytotoxic effects	Cell viability	30 μg/mL–48 h	Carvacrol exhibited a cytotoxic effect against gastric cancer cells	Maryam et al. (2015), Iran
	48 h of incubation
HL-60	10–200 μM	MTT assay	Apoptosis induction	Cell viability	HL-60–100 μM–24 h	Carvacrol effectively blocked the proliferation of cancer cells in vitro	Bhakkiyalakshmi et al. (2016), India
Jurkat	24 h of incubation		Cytotoxic effects	MMP	Jurkat - 50 μM–24 h
			Generation of ROS	Bcl-2
			Caspase-3
			Bax
Tca-8113	10–80 μM	—	Apoptosis induction	Cell proliferation	—	Carvacrol is a powerful new natural anti-cancer drug for human OSCC	Dai et al. (2016), China
SCC-25	24 and 48 h of incubation			S-Phase cells
			p21	CCND1
				CDK4
				Bcl-2
			Bax	MMP-2 and -9
				COX-2
A549	1–1,000 μM	SRB assay	Antiproliferative effects	—	A549–0.118 ± 0.0012 mΜ–72 h	Carvacrol exhibited antiproliferative and antioxidant effects. In addition, it exhibited more potent cytotoxicity against cells (A549). The cells (Hep3B) were more resistant to treatment and the cells (HepG2) were less sensitive	Fitsiou et al. (2016), Greece
HepG2	72 h of incubation		Cytotoxic effects		HepG2 - 0.344 ± 0.0035 mΜ–72 h
Hep3B					Hep3B- 0.234 ± 0.017 mΜ–72 h
PC-3	250–750 μM	CCK-8 Kit	—	Cell viability	PC-3 - 498.3 ± 12.2 μM–24 h	Carvacrol treatment suppresses cell proliferation, migration and invasion, indicating that it has antiprostatic effects in vitro	Luo et al. (2016), China
DU 145	24, 48 and 72 h of incubation			Cell proliferation	DU 145–430.6 ± 21.9 μM–24 h
				Cell migration
				Wound healing
				MMP-2
				PI3K/Akt and MAPK
				Cell invasion
				TRPM7
A549	0–250 μM	—	Cytotoxic effects	Cell viability	—	Carvacrol has cytotoxic activity	Coccimiglio et al. (2016), Canada
	24 h of incubation
U87	1–10,000 μM	MTT assay	Anticancer activity	—	U87–322 μM–24 h	Carvacrol exerted anticancer and antiproliferative activity with greater effect against the breast cancer cell line	Baranauskaite et al. (2017), Lithuania
MDA-MB 231	24 h of incubation		Antiproliferative activity		MDA-MB 231–199 μM–24 h
			Antioxidant activity
HepG2	0.01–0.25 μg/μL	MTT assay	—	Cell viability	48 mg/L–24 h	Carvacrol has therapeutic potential in tumor cells without adverse effects in healthy cells	Elshafie et al. (2017), Italy
	24 h of incubation			Hepatocarcinoma cells
PC-3	100–800 μM	—	Cytotoxic effects	Cell viability	—	Carvacrol is cytotoxic	Horng et al. (2017), China
	24 h of incubation
DU 145	10–500 μM	MTT assay	Cytotoxic effects	Cell viability	84.39 μM–24 h	Carvacrol has antiproliferative potential and can act as a chemopreventive agent in prostate cancer	Khan et al. (2017), India
	24 and 48 h of incubation		Apoptosis induction	Cell proliferation	42.06 μM–48 h
			Caspase-3	Mitochondrial membrane potential
			Generation of ROS	Cell cycle stop
			Cells in phase G0/G1	Cells in S and G2/M phases
SiHa	140–450 μM	MTT assay and LDH	Cytotoxic effects	Cell viability	SiHa - 424.22 μmol –24 h and 339.13 μmol–48 h	Carvacrol exhibited antiproliferative effects and may be a potential chemotherapeutic agent against cancer	Abbas and Al-Fatlawi (2018), Iraq
HepG2	24 and 48 h of incubation		Apoptosis induction	Bcl-2
			Caspase-3, -6 and -9		HepG2 - 576.52 μmol –24 h and 415.19 μmol –48 h
			Bax
			p53
A375	3.906–1,000 μg/mL	MTT assay	Apoptosis induction	Cell viability	40.41 ± 0.044 μg/mL–24 h	Carvacrol exhibits antiproliferative effects	Govindaraju and Arulselvi (2018), India
	24 of incubation		Sub-G1 phase	Cell growth
				Bcl-2
				Cell cycle stop
				Cells in phase G0/G1 and G2/M
AGS	10–600 µM	CellTiter-Glo Luminescent cell viability assay	Apoptotic effects	Cell viability	82.57 ± 5.58 µM–24 h	Carvacrol has cytotoxic effects, apoptotic, genotoxic effects and dose-dependent ROS generators	Günes-Bayir et al. (2018), Turkey
	24 h of incubation		Necrosis	Bcl-2
			Bax
			Caspase-3 and -9
			Generation of ROS	GSH levels
			Genotoxic effect
AGS	10–600 µM	CellTiter-Glo Luminescent cell viability assay	Cytotoxic effects	Cell viability	82.57 ± 5.5 μM–48 h	Carvacrol inhibited cell proliferation and induced cytotoxicity in cancer cells	Günes-Bayir et al. (2018), Turkey
	48 h of incubation		Apoptosis induction	Bcl-2
			Bax
			Caspase-3 e -9
			Generation of ROS	GSH levels
			Genotoxic effect
MCF-7	10–200 μg/mL	MTT assay	—	Cell viability	MCF-7 - 46.5 μg/mL–24 h	Carvacrol has a cytotoxic effect and can cause inhibition of cell growth	Jamali et al. (2018), Iran
MDA-MB 231	24 h of incubation				MDA-MB 231–53 μg/mL– 24 h
A549	30–300 μM	—	—	Cell viability	-	Carvacrol suppressed cell proliferation and migration and its inhibitory effect was attenuated in NSCLC cells with overexpression of AXL	Jung et al. (2018), Republic of Korea
H460	24 h of incubation			Cell proliferation
				AXL expression
				Cell migration
JAR	50–300 μM	—	Apoptosis induction	Cell proliferation	—	Carvacrol may be a possible new therapeutic agent or supplement for the control of human choriocarcinomas	Lim et al. (2019), Republic of Korea
JEG3	48 h of incubation		Sub-G1 phase	Cell viability
			Generation of ROS	PI3K/AKT
			p-JNK	p-ERK1/2
			p-p38	MMP
HeLa	100–800 µM	XTT Reduction assay	Induction of cytotoxicity and apoptosis	Cyclin D1	556 ± 39 μM–24 h	Carvacrol can be used to treat cervical cancer, however, it should be avoided during cisplatin chemotherapy	Potočnjak et al. (2018), Croatia
	24 h of incubation		ERK1/2
			Caspase-9
			p21
PC-3	100–800 μM	MTT assay	Cell death	Cell viability	360 μM–48 h	Carvacrol inhibited the ability to invade and migrate PC3 cells and can be considered an anticancer agent	Heidarian and Keloushadi (2019), Iran
	48 h of incubation			Cell proliferation
				Tumor cell invasion
				IL-6
				p-STAT3
				p-ERK1/2
				p-AKT
PC-3	10–500 μM	MTT assay	Apoptosis induction	Cell viability	46.71 μM–24 h	Carvacrol is a chemopreventive agent and has an antiproliferative effect on prostate cancer cells	Khan et al. (2019), India
			Caspases -8 e -9	Cell proliferation
				Cell migration
	24 and 48 h of incubation		Generation of ROS	Cell cycle stop at G0/G1	39.81 μM–48 h
				Cells in S and G2/M phases Bcl-2
			Bax	Notch-1
				mRNA Jagged-1
MCF-7	31.2–500 μg/mL	AlamarBlue® assay	Apoptosis induction	Cell proliferation	MCF-7 - 266.8 μg/mL–48 h	Carvacrol had the most cytotoxic effect among the other components studied	Tayarani-Najaran et al. (2019), Iran
PC-3	48 h of incubation		Cytotoxic effects	Cell viability	PC-3 - >500 μg/mL– 48 h
DU 145			Bax		DU 145–21.11 μg/mL– 48 h
PC-3	25–200 μg/mL	—	Cytotoxic effects	Cell viability	—	At the lowest concentration tested (25 μg/ml), carvacrol did not exhibit cytotoxicity to cancer cells	Trindade et al. (2019), Brazil
	24 and 48 h of incubation
HCT116	25–200 μM	xCELLigence Real-time cell Analysis	—	Cell proliferation	HCT116–92 μM–48 h	Carvacrol has an antiproliferative effect on both cell lines, but is more efficient against HT-29 compared to the HCT116 cell line	Pakdemirli et al. (2020), Turkey
HT-29	48 h of incubation				HT-29–42 μM–48 h
MCF-7	25–250 μmol/L	MTT and LDH assay	Apoptosis induction	Cell viability	200 μmol/L–24/48 h	Carvacrol can be used in a new approach for the treatment of breast cancer	Mari et al. (2020), India
			Cells in phase G0/G1	Cells in S and G2 phase
				CDK4 and 6
	24 and 48 of incubation			Cyclin D1
			Bax	Bcl-2
				PI3K/p-AKT
SKOV-3	100, 200, 400, 600 μM	MTT assay	Apoptosis induction	Cell viability	322.50 µM–24 h	Carvacrol was cytotoxic to the ovarian cancer cell line	Elbe et al. (2020), Turkey
	24 and 48 h of incubation				289.54 µM–48 h
Kelly	12.5, 25, 50 µM	—	Antiproliferative effects	—	—	Carvacrol can be used to inhibit neuroblastoma cell proliferation	Kocal and Pakdemirli (2020), Turkey
SH-SY5Y	24 h of incubation
BT-483	25–500 μM	—	Apoptosis induction	Cell viability	—	Carvacrol suppresses breast cancer cells by regulating the cell cycle and the TRPM7 pathway is one of the pharmacological mechanisms	Li et al. (2021), China
BT-474			Cells in G1/G0 phase	S-phase and G2/M cells
MCF-7	24 h of incubation		Cyclin C, D and E	Cell proliferation
MDA-MB 231				Cyclin A e B
MDA-MB 453				CDK 4
KG1	100, 200, 300, 400 μM	—	Cell death	Cell viability	—	KG1 cell lines were very sensitive to 300 µM carvacrol compared to the HL60 line, while the K562 line showed resistance after 48 h of treatment with 400 µM carvacrol	Bouhtit et al. (2021), Belgium
K-562	24 and 48 h of incubation
HL-60

Model	Dose	Results/targets		Conclusion	Authors (Year), Country
		Increase	Decrease
In vivo studies
Chemical carcinogenesis induced by B [a]P in male wistar rats	20 mL of carvacrol (ε = 976 mg/mL) mixed with 200mg of B [a]P	Anticarcinogenic effects	30% tumor incidence	Carvacrol has anticarcinogenic, antiproliferative and antiplatelet properties	Karkabounas et al. (2006), Greece
		Animal survival time	B [a]P carcinogenic potency
Male wistar rats induced with hepatocarcinogenesis providing 0.01% DEN through drinking water for 16 weeks	Pre-treatment: (15 mg/kg b.wt) of carvacrol orally one week before DEN administration and up to 16 weeks	Final body weight	Pre-treatment: Number of nodules; neoplastic transformations; liver weight	Carvacrol has the ability to cause apoptosis in cancer cells and has a potent elimination of free radicals and antioxidant activities	Jayakumar et al. (2012), India
	Post-treatment: (15 mg/kg b.wt) of carvacrol orally for 6 weeks after administration of DEN for 10 weeks	Chemopreventive effect apoptosis induction	Post-treatment: Persistent but tiny nodules; architecture;
		GPx, GR, GSH, SOD, CAT	Likely to spread through intrahepatic veins
			AST, ALT, ALP, LDH, cGT
Liver carcinogenesis chemically induced by NDEA in male wistar rats orally (dissolved in 0.9% normal saline), in a dose of 20 mg/kg body weight, five times a week, for 6 weeks	15 mg/kg of carvacrol orally, five times a week for 15 weeks, after NDEA administration for 6 weeks	Antiproliferative effect	AFP	Carvacrol may have an antitumor effect through its antiangiogenic capacity, antiproliferative effect and apoptotic activity against tumor cells in vivo	Ahmed et al. (2013), Egypt
		Apoptosis induction	VEGF
		Marked improvement in histological characteristic of liver tissue	AFU
			GGT
Male wistar rats induced with hepatocarcinogenesis providing 0.01% DEN through drinking water for 16 weeks	Pre-treatment: (15 mg/kg b.wt) of carvacrol orally one week before DEN administration and up to 16 weeks	—	Cell proliferation	Carvacrol attenuates hepatocellular carcinoma by inhibiting cell proliferation and tumor metastases	Subramaniyan et al. (2014), India
	Post-treatment: (15 mg/kg b.wt) of carvacrol orally for 6 weeks after administration of DEN for 10 weeks		Tumor markers
			Mast cell density
			PCNA
			MMP-2 and -9
			AgNORs
DMH-induced colon carcinogenesis in male Wistar rats who received subcutaneous injections of DMH (20 mg/kg b.wt) in the right thigh, once a week for the first 4 weeks of the experiment (four injections)	20, 40 or 80 mg/kg every day from the day of the carcinogen treatment till the end of the 16th week	Weight gain	Incidence of tumors	Carvacrol has antiproliferative, anticarcinogenic and chemopreventive potential and its effects were better observed at a dose of 40 mg/kg b.wt	Sivaranjani et al. (2016), India
		Growth rate	Growth of neoplastic polyps
		GPx, GR, GSH, SOD, CAT
C57BL/6 mice induced with DEN hepatocellular carcinoma injected intraperitoneally at a dose of 100 mg/kg	—	Intrastromal and peritumor lymphocytes	Tumor growth	The direct regulation relationship between DAPK1 and PPP2R2A may be the biological mechanism of tumorigenesis and progression of hepatocellular carcinoma	Li et al. (2019), China
	Intragastrically	DAPK1	Tumor cells
	20 weeks		Mitotic phase
			PPP2R2A
DMBA-induced breast cancer in female Holztman mice in a single administration of DMBA by oral gavage at a dose of 80 mg /kg body weight	50, 100 and 200 mg/kg/day	Tumor latency	Number of tumors	Carvacrol had an antitumor effect on breast cancer in vivo and it is likely that this effect may be due to its antioxidant activity	Rojas-Armas et al. (2020), Peru
	Oral gavage		75% in the frequency of tumors
	14 weeks		67% in the incidence of tumors
			Average volume